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Description
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On December 28 and 29 2019, we obtained 17 male and 17 female last-level (counting from soil level) cladodes which length ranged from 19 through 25 cm, from individuals identified and tagged by their sexual form in our previous studies. The collected cladodes were less than eleven months old, that is, they grew from buds produced by the mother cladode in the spring of the same season. To estimate the effect of arbuscular micorrhizal fungi (AMF) on plant growth traits, we planted the collected cladodes under greenhouse conditions, without temperature control (4 January 2020 to 30 October 2020). We disinfected them with a 5% sodium hypochlorite solution, dried at room temperature and planted in the substrate composed by agrolite (Accimin), pittmos (Better Homes & Gardens) and soil obtained directly from the root system of the sampled plants from which the cladodes were obtained. All the material we used for the preparation of the substrate was treated with cobalt-60 gamma rays at the National Institute of Nuclear Research (ININ = Instituto Nacional de Investigaciones Nucleares), at a dose of 11.22 through 13.57 kGy (Rodríguez Suarez, 2001). The accuracy of irradiation within the limits required by our experiment was confirmed by the certificate No. IR2019005022 released by ININ. Typically, gamma-irradiation at 10 kGy eliminates all fungi (McNamara et al., 2003). Afterwards, we planted the cladodes individually in 13.6 L black plastic bags (35 × 21 × 18.5 cm) which were 4/6 filled with the volumetric mixture in a 2 : 5 : 2 ratio of agrolite, pittmos and soil (sieved through a 5 mm sieve). To quantify the number of spores present in non-radiatet soil, we used the wet sieving technique (Gerdemann & Nicolson, 1963). We use 300 µm, 180 µm, 75 µm, 63 µm, and 38 µm sieves. The remains of the last two sieves were collected and centrifuged at 3000 rpm for 10 min, in 50 ml falco tubes with 15 ml of sucrose solution (50% w/v) and 10 ml of the samples obtained from the sieves. We decanted the supernatant on filter paper and rinsed with distilled water. We performed the spore count with an optical microscope (Olympus CX31) at 10X. We assigned the collected cladodes (called “mother cladodes”) to two experimental groups: the first one formed with 12 male and 12 female cladodes grown on the non-sterilised substrate, with soil containing 500 AMF spores per 100 g of soil (= colonised group), and the second one, composed by five male and five female cladodes, grown on the fully sterilised substrate (without AMF spores = control group). We tagged all cladodes by sex and experimental group. We placed 21cm x 18.5 cm x 35 cm (volume = 13.6 l) polyethylene pots with plants arranged as in a completely random block on cement floor in a greenhouse built specifically for this study (2.5 m × 1.5 m × 1.5 m (high)), with natural light and darkness period and a temperature ranging from 19-21°C (night) to 23-27°C (day). We grew the cladodes from January 4 to October 30, 2020. We watered all cladodes every 30 days with 200 ml of purified water (electropure) and estimated the relative growth rate (RGR) of each daughter cladode by measuring the length, width and thickness to the nearest 1 mm every 30 days using a Pretul calliper. At the end of the experiment, we randomly harvested seven males and seven females from the colonised group and three males and three females from the control group. We counted the number of roots produced, measured their length and thickness with a Pretul calliper, and weighed their roots using a Mainstays digital scale (WD61477). We also quantified the number of daughter cladodes produced from each sexual form of each experimental group and weighed them individually on a Mainsteys digital scale (WD61477). (2024-08-30)
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